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A Combination of NMR Methods to Reveal the Interfacial Structure of a Pharmaceutical Nanocrystal and Nanococrystal in the Suspended State Taro Kojima, Masatoshi Karashima, Katsuhiko Yamamoto, and Yukihiro Ikeda Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00360 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Molecular Pharmaceutics

1

A Combination of NMR Methods to Reveal the

2

Interfacial Structure of a Pharmaceutical

3

Nanocrystal and Nanococrystal in the Suspended

4

State

5

Taro Kojima*, Masatoshi Karashima, Katsuhiko Yamamoto, Yukihiro Ikeda

6

Analytical Development, Pharmaceutical Sciences, Takeda Pharmaceutical Company Limited

7

26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan

8

ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

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ABSTRACT. The detailed structure of a pharmaceutical nanosuspension was investigated using

2

three NMR methods: solid-state, solution-state, and high resolution-magic angle spinning (HR-

3

MAS) NMR. Carbamazepine (CBZ) and CBZ-saccharin (SAC) cocrystal nanosuspensions were

4

prepared by wet-milling with hydroxypropyl methylcellulose (HPMC) and sodium dodecyl

5

sulfate (SDS) as stabilizing agents. Solid-state

6

crystalline drug substance but also solid-state HPMC, even though HPMC was used as an

7

aqueous solution to prepare the nanosuspensions. Solution-state 1H NMR of the nanosuspensions

8

with and without ultracentrifugation pre-treatment indicated that a fraction of the CBZ, SAC, and

9

SDS formed a solid or semi-solid phase on the surface of the nanoparticles and was in

10

equilibrium between the dissolved and undissolved states. 1H HR-MAS NMR was highly

11

effective in detecting and quantifying the semi-solid phase on the surface of the nanoparticles.

12

From these comprehensive NMR studies, it was concluded that the nanosuspension was

13

composed of crystalline drug core particles surrounded by a semi-solid phase consisting of the

14

drug and stabilizing agents. The semi-solid phase on the nanoparticle surface was in equilibrium

15

with the solution phase and contributed to the stabilization of the nanoparticle by steric hindrance

16

and electrostatic repulsion.

17

KEYWORDS. nanocrystal, nanococrystal, cocrystal, interfacial structure, solid-state NMR,

18

solution-state NMR, high-resolution NMR, molecular mobility

13

C NMR indicated the presence of not only the

19


2

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INTRODUCTION. Nanosuspension formulation is a promising method to improve the oral

2

bioavailability of poorly water-soluble drugs, as the significantly increased surface area of the

3

nanoparticles leads to a rapid dissolution rate.1-4 However, nanosuspensions face the inherent

4

challenge of avoiding aggregation and agglomeration of the nanoparticles.5 The extreme increase

5

in the surface area is connected to a significant increase in the free enthalpy resulting in a

6

thermodynamic driving force favoring a situation where the surface area is again reduced to a

7

minimum.6 Because of this thermodynamic behavior, stabilizing agents are usually used to

8

prevent nanoparticle agglomeration.7 However, the development of nanoparticle formulations is

9

practically limited to a trial and error approach, as it is still unclear which stabilizers are suitable,

10

and what amount of stabilizer is optimal for the nanosuspensions. Therefore, understanding the

11

molecular interactions among drugs and stabilizers in the nanosuspensions is crucial for the

12

efficient design of nanosuspension formulations. Although there has been a great deal of

13

discussion over the term, a cocrystal is defined as a crystal that consists of two or more

14

molecular species held together by non-covalent forces.8-12 Cocrystals have attracted great

15

attention in the pharmaceutical industry due to the many benefits of their advantageous

16

physicochemical properties in terms of solubility, dissolution, melting point, and physical and

17

chemical stability compared to single component crystals.8, 10, 13-16 Hence, cocrystals are also

18

used to enhance the oral bioavailability of poorly water-soluble drugs. In a previous study, we

19

successfully developed a novel formulation combining nanosuspension and cocrystal

20

technologies, aiming to achieve a synergistic effect between nanonization and cocrystallization.17

21

Carbamazepine (CBZ) and carbamazepine-saccharin cocrystal (CBZ-SAC) nanosuspensions

22

were prepared using a wet milling method, and their physicochemical properties were

23

investigated. The CBZ-SAC nanosuspension was found to have a faster dissolution rate as well


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Page 4 of 29

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as enhanced solubility. However, although both the CBZ and CBZ-SAC nanosuspensions were

2

prepared by the same method and using the same stabilizers, some differences were observed

3

between their physicochemical properties. The previous study has provided us with the

4

opportunity to obtain novel insights into nanosuspensions by determining their precise structures

5

at the molecular level.

6

NMR spectroscopy is a powerful tool that provides detailed information at the molecular level,

7

regardless of the sample conditions. Conventional solution-state NMR and solid-state NMR

8

techniques have been widely used in various fields.18, 19 In addition, suspended-state NMR or in-

9

situ NMR, a technique in which solid-state NMR is applied to samples containing a mixture of

10

solid and dissolved components, is also often used to investigate a whole suspension sample

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without preprocessing, and has clarified some of the unique features of nanosuspensions.20-22 In a

12

study of nanoparticles in pharmaceutical applications, Zhang et al. observed solid-liquid

13

exchange between probucol and the polyvinylpyrrolidone/sodium dodecyl sulfate complex at the

14

surface of the nanoparticle by

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investigated piroxicam/poloxamer nanoparticles using suspended-state NMR, and reported that

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piroxicam nanocrystals formed a core-shell structure with crystalline and amorphous piroxicam

17

surrounded by poloxamer chains.24 Although these studies successfully determined the interfacial

18

structure of the nanoparticles, directly observing the surface structure of the nanoparticles was

19

difficult. The surface structures of the amorphous drug and polymeric stabilizing agents were

20

thought to be in the intermediate mobility region between solid and solution; hence, neither

21

solid- nor solution-state NMR was able to detect the signals from the surface structure. The

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combination of the conventional solution-state NMR approach with magic angle spinning (MAS)

23

NMR, which is called high-resolution (HR)-MAS NMR, was developed several decades ago to

13

C solution-state NMR measurement.23 Hasegawa et al.


4

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1

bridge the gap between solid- and solution-state NMR.25-27 The HR-MAS NMR technique has

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been applied to various types of samples such as biomolecules,28, 29 food materials,30 and lipid

3

membranes.31 HR-MAS NMR is also applied to pharmaceutical formulations in order to detect

4

drug molecules in viscous solutions, creams, gels, and pastes.26, 32 However, despite its potential

5

capability to provide novel insights into the interfacial structure of nanoparticles, there are only a

6

few reports regarding the use of HR-MAS NMR which analyze the surface structure of

7

pharmaceutical nanosuspensions. In the present study, we performed a comprehensive NMR

8

study, including HR-MAS NMR, to elucidate the structure of the CBZ and CBZ-SAC

9

nanosuspensions in detail. We applied a multi-perspective analysis of the NMR measurements

10

based on the molecular mobility approach to determine the nanosuspension structure. We also

11

discuss the physicochemical properties and stabilization mechanism of the nanosuspensions.

12

MATERIALS AND METHODS

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Materials. Anhydrous carbamazepine (CBZ, M.W. 236.27), saccharin (SAC, M.W. 183.18), and

14

sodium dodecyl sulfate (SDS, M.W. 288.38) were purchased from Wako Pure Chemical

15

Industries, Ltd. (Osaka, Japan). Hydroxypropyl methylcellulose (HPMC, TC-5E grade) was

16

provided by Shin-Etsu Chemical, Co., Ltd. (Tokyo, Japan). The molecular structures of CBZ,

17

SAC, HPMC, and SDS are shown in Figure 1. Deuterium oxide and 3-(trimethylsilyl)propionic-

18

2,2,3,3-d4 acid sodium (TSP-d4) were also purchased from Wako Pure Chemical Industries, Ltd.

19

Hexamethylbenzene (HMB) was purchased from Acros Organics (Geel, Belgium). All materials

20

were used as received. CBZ dihydrate was prepared as a slurry of 1 g of anhydrous CBZ in 75

21

mL distilled water at room temperature overnight. The CBZ-SAC cocrystal (CBZ-SAC) was also

22

prepared by a slurry method. Equimolar amounts of CBZ and SAC were suspended in

23

acetonitrile and stirred at room temperature overnight. The precipitate was collected by vacuum


5


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1

filtration and dried under vacuum at room temperature overnight. The formation of CBZ

2

dihydrate and CBZ-SAC was confirmed by powder X-ray diffraction (PXRD) using an Ultima

3

IV (Rigaku Corporation, Japan)

4 5

Figure 1. The molecular structures of CBZ, SAC, HPMC, and SDS.

6 7

Preparation of Nanosuspensions. The nanosuspensions were prepared according to the method

8

described in the previous study using CBZ and CBZ-SAC as model active pharmaceutical

9

ingredients (APIs).17 Briefly, 2% (w/v) API was dispersed into 5 mL of the formulation vehicle,

10

which consisted of 0.5% (w/v) HPMC and 0.02% (w/v) SDS in distilled water. The API

11

dispersion was wet-milled by using a NP-100 rotation/revolution nano pulverizer (Thinky Co.,

12

Ltd., Japan) with 10 g of 0.1 mmφ zirconia beads. 20% (w/v) API nanosuspensions were also

13

prepared to compensate for the low sensitivity of the solid-state NMR measurement; 20% (w/v)


6

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1

API, and 5 mL distilled water containing 5% (w/v) HPMC and 0.2% (w/v) SDS solution were

2

used for the same milling process.

3 4

Particle Size Distribution and Zeta Potential. The particle size distribution and zeta potential

5

of the nanosuspensions were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd.,

6

UK). The nanosuspensions were diluted with distilled water and used for the measurement of

7

particle size and zeta potential. The polydispersity index (PDI) was also measured to evaluate the

8

size distribution of the nanosuspensions.

9 10

Ultracentrifugation

11

Nanosuspensions. The nanosuspensions were centrifuged at 150000 ×g for 40 min using an

12

Optimax Ultracentrifuge with an MLA-130 rotor (Beckman Coulter, USA). The supernatant was

13

used for NMR analysis. The residual solid was re-suspended in water for use in particle size and

14

zeta potential measurements to confirm the maintenance of the nanoparticle structure.

to

Separate

the

Solution

and

Solid

Components

in

the

15 16

Concentration of CBZ in Water and the Formulation Vehicle. CBZ dihydrate and CBZ-SAC

17

were suspended in distilled water and the formulation vehicle, followed by incubation at 25°C

18

for 18 hours. The obtained suspensions were filtered using 0.20 µm membrane filters. The CBZ

19

in the filtrate was quantitated using an HPLC method. The experiment was performed in

20

triplicate.

21 22

NMR Spectroscopy. All the NMR experiments were performed using a JNM-ECX500II (JEOL

23

RESONANCE, Japan) with a magnetic field strength of 11.7 T. A 3.2 mm HX MAS probe


7


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1

sHX32 (JEOL RESONANCE, Japan) was used for the solid-state NMR experiments.

2

Approximately 60 mg or 40 µL of the sample was placed into a 3.2 mmφ zirconium rotor and

3

sealed with a vespel® cap.

4

spectra were acquired with high-power 1H decoupling at a MAS frequency of 6 kHz with an inlet

5

air temperature of 25°C. A total sideband suppression (TOSS) sequence was utilized to suppress

6

the spinning side bands. A total accumulation number of up to 616 was used for the solid

7

materials depending on the relaxation delay time, while a value of 28006 was used for the

8

nanosuspensions because of their low solid content. The relaxation delay time was set based on

9

the 1H spin-lattice relaxation time (T1H) of the material, which was measured using a standard

10

inversion recovery or saturation recovery method (Table S1 in supporting information). A

11

relaxation delay of more than 1.3 times the T1H was used for efficient data accumulation. A fixed

12

value of 120 s was used for those samples that had a T1H longer than 90 s. Specifically, the

13

relaxation delay was 3 s for HPMC and SDS, 32 s for CBZ dihydrate, and 120 s for CBZ

14

anhydrate, SAC, and CBZ-SAC. A relaxation time of 3 s was used for the nanosuspensions to

15

emphasize the signal from the high molecular mobility region, which was thought to represent

16

minor components in terms of the mass ratio. A CP contact time of 6 ms was used, except for in

17

the cases of SAC, HPMC, SDS, for which a contact time of 3 ms was used. The 1H 90° pulse

18

was calibrated using HMB and was typically 2.68 µs. All spectra were externally referenced to

19

the methine peak of HMB at 17.2 ppm.

13

C cross polarization and magic angle spinning (CP/MAS) NMR

20

The conventional solution-state NMR experiments and high resolution and magic angle

21

spinning (HR-MAS) NMR measurements were performed using a 5.0 mm ROYAL probe

22

RO5AT/FGSQ (JEOL RESONANCE, Japan) and a 3.2 mm FG/MAS probe sCH32 (JEOL

23

RESONANCE, Japan), respectively. D2O containing 0.5% (w/v) TSP-d4 was added to the


8

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1

sample solutions and suspensions at a ratio of 10% (v/v) to provide a signal for the magnetic

2

field frequency lock and internal chemical shift standard. The methyl proton peak of TSP-d4 was

3

set to 0 ppm. This signal was also used as an internal reference standard for quantitative analysis.

4

In the quantitative analysis, the following signals were integrated: CBZ, aliphatic protons at 6.94

5

ppm; SAC, aromatic protons between 7.70-8.00 ppm; HPMC, methyl protons at 1.17 ppm; SDS,

6

methyl protons at 0.87 ppm. The sample suspensions and solutions were transferred into a 5

7

mmφ glass sample tube or a 3.2 mm zirconium rotor with a Kel-F® cap. The NMR spectra were

8

recorded with an inlet air temperature of 25°C. The HR-MAS NMR spectra were acquired at a

9

MAS frequency of 5 kHz. The Watergate sequence was utilized to suppress the signal of the

10

solvent H2O. A 1H 90° pulse of 7.3 µs for conventional solution-state NMR and 4.15 µs for HR-

11

MAS NMR were used. A relaxation delay time of 30 s was applied for all samples to ensure

12

complete magnetization recovery; this time was longer than five times the T1H of all the peaks

13

used for integration (Table S2 in supporting information). The accumulation numbers were set to

14

16-512 to obtain an appropriate signal to noise ratio.

15 16

RESULTS AND DISCUSSION

17

Preparation of Nanosuspensions. The particle size, PDI, and zeta potential of the

18

nanosuspensions are presented in Table 1. The nanosuspensions were successfully prepared as

19

reported. However, the 20% (w/v) API formulation of the CBZ-SAC nanosuspension was too

20

unstable against dilution with distilled water to carry out the particle size measurement

21

appropriately, as the initially transparent dilution became cloudy within one minute. It was

22

assumed that the increased viscosity of the vehicle solution reduced the milling efficiency and

23

resulted in incomplete nanosuspension formation. Therefore, the 20% (w/v) formulation of CBZ-


9


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1

SAC was prepared by concentrating the 2% (w/v) nanosuspension to 20% (w/v) by

2

ultracentrifugation, followed by reconstitution using the formulation vehicle, which consisted of

3

5% (w/v) HPMC and 0.2% (w/v) SDS in distilled water. The reconstituted 20% (w/v) CBZ-SAC

4

suspension was nano-sized, although a slight increase in the particle size and PDI were observed.

5

These nanosuspensions were used for the following NMR studies.

6

Table 1. Particle Size Distribution and Zeta Potential of CBZ and CBZ-SAC Nanosuspensions

API (content)

Particle size (d50, nm)

PDI

Zeta potential (mV)

CBZ (2% (w/v))

230.5

0.167

-21.0

CBZ (20% (w/v))

191.7

0.160

-22.7

CBZ-SAC (2% (w/v))

296.4

0.163

-5.6

CBZ-SAC (20% (w/v))

347.8

0.235

-2.8

7 13

8

Solid-state NMR Measurements. Solid-state

9

out to evaluate the solid components in the nanosuspensions using 20% (w/v) API. The effect of

10

MAS on the sample suspensions structure should be taken into account before discussing the

11

results when MAS is applied to a suspended sample, as the strong centrifugal forces induced

12

during the measurement may result in the sedimentation and potential structure changes of the

13

sample. Although sediment was present inside the sample tube after the MAS measurements, a

14

homogeneous suspension was formed by simple resuspension of the sediment. The particle size

15

of the re-dispersion of the CBZ and CBZ-SAC nanosuspensions after the MAS measurements

16

was 186.7 and 313.1 nm, respectively (Table S3 in supporting information). The re-dispersible

17

nature of the sedimentary sample implied that the MAS had no significant effect on the

18

nanoparticle structure. Figure 2 shows the 13C CP/MAS NMR spectra of the CBZ and CBZ-SAC

19

nanosuspensions. The

13

C CP/MAS NMR measurements were carried

C CP/MAS NMR spectra of the CBZ and CBZ-SAC nanosuspensions


10

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1

were almost identical to those of solid CBZ dihydrate and CBZ-SAC, respectively. This result

2

indicated that the major solid component in each nanosuspension was the crystalline API,

3

because the CP process involves magnetization transfer via dipolar interaction.33 Hence,

4

CP/MAS NMR is active for low molecular mobility components such as crystalline solids, but is

5

practically inactive for high molecular mobility components such as solvents and dissolved

6

molecules.6, 34

7

CBZ anhydrate to the CBZ dihydrate. As shown in Figure 2 - (a) and - (b), the

8

NMR measurement was able to distinguish the molecular coordination of CBZ anhydrate and

9

dihydrate in their crystal structures. In our previous study, it was confirmed by PXRD

10

measurements that CBZ dihydrate was formed during the wet milling process.17 However NMR

11

measurements can be more suitable for suspended samples than PXRD measurements, as PXRD

12

measurements require sample preprocessing to obtain dry samples, which may cause structural

13

changes to the nanoparticle. Focusing on the 50-110 ppm region, it should be emphasized that

14

small peaks derived from HPMC were detected in the CBZ and CBZ-SAC nanosuspensions. The

15

HPMC peaks in

16

HPMC in the nanosuspensions, even though HPMC was used as a solution to prepare the

17

nanosuspensions. This result strongly suggested that solidification of HPMC occurred during the

18

wet milling process. The wet milling process drove both the breakdown of the crystalline API

19

and the adsorption of HPMC onto the crystalline surface. This simultaneous breakdown and solid

20

phase formation process was considered to be important in promoting nanonization, because the

21

newly created API crystal surfaces were hydrophobic, and water molecules would be

22

energetically driven to leave.5 Thus, the solid HPMC phase on the new crystal surfaces would

23

help to prevent agglomeration of the nanoparticles by increasing their hydrophilic affinity with

13

13

C

C CP/MAS NMR measurement also showed crystal form transition from the

13

13

C CP/MAS

C CP/MAS NMR spectra clearly demonstrated the presence of solid-state


11


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1

the aqueous medium. SDS was not detected in the 13C CP/MAS NMR spectra. Its absence from

2

the spectra was attributed to the low SDS content of the suspensions; even if all the SDS

3

molecules existed in the solid state, they would still be undetectable by solid-state NMR. From

4

these results, the solid composition detected in the nanosuspension was concluded to consist of

5

crystalline API particles and HPMC. It was assumed that HPMC solidified on the surface of the

6

crystalline nanoparticles during the wet milling process and helped to stabilize the

7

nanosuspensions.

8 9

Figure 2. Solid-state 13C CP/MAS NMR spectra of (a) CBZ anhydrate, (b) CBZ dihydrate, (c)

10

CBZ nanosuspension, (d) CBZ-SAC, (e) CBZ-SAC nanosuspension, (f) SAC, (g) HPMC, and

11

(h) SDS. The spectral region from 50 to 110 ppm were magnified and stacked upon each

12

spectrum except for (g) and (h). The white circles show solid HPMC resonances.


12

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1 2

Solution-state NMR Measurements. Solution 1H NMR spectra were acquired to investigate the

3

dissolved components in the nanosuspensions. Figure 3 shows the solution 1H NMR spectra of

4

the CBZ and CBZ-SAC nanosuspensions, and Figure 4 shows a scaled-up expansion of the

5

aliphatic and aromatic regions in Figure 3. In the solution-state, the CBZ, SAC, and SDS peaks

6

were sharp and well resolved, reflecting their high molecular mobility in the dissolved state

7

(Figures 3 - (a), (c), and (f)). The HPMC peaks in Figure 3 - (e) were broad due to the restricted

8

molecular mobility of this high molecular weight polymer structure. However, CBZ and SDS

9

were not shown in the spectra of the nanosuspensions (Figures 4 - (b) and (d)), even though the

10

solubility of CBZ and SDS were sufficient to allow their detection in solution 1H NMR (Figures

11

3 - (a) and (f)). Theoretically, solution-state NMR detects high molecular mobility components

12

such as dissolved molecules. Fejzo et al. reported that line broadening of the signal of a small-

13

molecule ligand was observed when the ligand was bound to a large-molecule protein because of

14

the equilibrium between the free and bound state.35 An exchange rate faster than the solution-

15

state NMR time scale results in averaging of the molecular mobility of the free and bound state

16

ligands, while in contrast, a slow exchange rate makes it possible to distinguish the bound and

17

unbound molecules as different resonances. Henoumont et al. also observed the pH dependent

18

line width increase and intensity decay, and ultimate disappearance, of the peptide signals in an

19

iron oxide nanoparticle system.36 The change in the line width and disappearance of the signals

20

were explained by the adsorption of the peptide onto the nanoparticle surface due to the

21

protonation of the amine moiety. The protonation enhanced the ionic bonding between the amine

22

moiety of the peptide and the carboxylate groups on the nanoparticle surface and led to

23

immobilization of the peptide molecule. These two studies suggest that if the molecular mobility


13


Page 14 of 29

1

of the absorbing phase was outside the motional regime of solution NMR, and the exchange rate

2

was faster than the NMR time scale, the signal from the unbound molecule could apparently

3

disappear from the solution-state NMR spectra due to the averaging effect on the molecular

4

mobility. Therefore, the lack of the CBZ and SDS peaks and the line broadening of the SAC

5

peaks strongly suggested an exchange between the solution and solid phase. It should be noted

6

that the increased viscosity of the formulation vehicle, 0.5% (w/v) HPMC and 0.02% (w/v) SDS

7

in water, did not have any impact on the linewidth of the CBZ, SAC, or SDS peaks (Figure S1 in

8

supporting information). Next, we performed ultracentrifugation to verify the hypothesis that the

9

lack of CBZ- and SDS-resonances was not due to the low solubility of these components but was

10

indicative of exchange-driven broadening.

11


14

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1

Figure 3. Solution 1H NMR spectra of (a) CBZ in water, (b) CBZ nanosuspension, (c) CBZ-SAC

2

in water (d) CBZ-SAC nanosuspension, (e) 0.5% (w/v) HPMC in water, and (f) 0.02% (w/v)

3

SDS in water. The arrows show the absence of CBZ and SDS peaks in the nanosuspensions.

4

5 6

Figure 4. Scaled-up expansion of solution 1H NMR spectra in aliphatic and aromatic regions: (a)

7

CBZ in water, (b) CBZ nanosuspension, (c) CBZ-SAC in water (d) CBZ-SAC nanosuspension,

8

(e) 0.5% (w/v) HPMC in water, and (f) 0.02% (w/v) SDS in water.

9

The CBZ and CBZ-SAC nanosuspensions were physically separated into the solid and

10

solution phase by ultracentrifugation to focus on the dissolved components. The solution 1H

11

NMR spectra of the supernatants are shown in Figure 5. Interestingly, CBZ and SDS peaks were

12

clearly detected in the solution 1H NMR spectra of the supernatants, unlike in the spectra of the

13

un-centrifuged samples shown in Figure 3. Sharp and resolved SAC peaks were also detected in

14

the supernatant. The free fractions of CBZ, SDS, and SAC were detected as a result of their


15


Page 16 of 29

1

original high molecular mobility state being restored by the elimination of the solid phase, which

2

reduced their apparent molecular mobility due to equilibrium. This result supported the proposed

3

exchange between the free and bound state in the nanosuspensions. It is also important to

4

consider that the exchange should occur on the surface of the nanoparticle, since the equilibrium

5

was an interfacial reaction between two phases, in this case the solution and solid phases. The

6

interfacial structure of the nanoparticle was assumed to be composed of a mixture of the API and

7

stabilizing agents and was flexible enough, i.e., a semi-solid phase, to undergo rapid exchange

8

with the solution phase.

9 10

Figure 5. Solution 1H NMR spectra of supernatants of (a) CBZ nanosuspension, (b) CBZ-SAC

11

nanosuspension, and (c) CBZ and SAC in the formulation vehicle of 0.5% (w/v) HPMC and

12

0.02% (w/v) SDS in water. Each spectrum was magnified individually to compare the spectra.

13 14

HR-MAS NMR Measurements. 1H HR-MAS NMR measurements of the CBZ and CBZ-SAC

15

nanosuspensions were conducted to directly evaluate the interfacial structure, which was thought


16

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

to include a semi-solid phase showing moderate molecular mobility. Figure 6 shows the 1H HR-

2

MAS NMR spectra of the CBZ and CBZ-SAC nanosuspensions. Importantly, CBZ, SAC, and

3

SDS in the nanosuspensions (Figures 6 - (a) and (b)) were slightly broader than those in the

4

reference spectrum (Figure 6 - (c)). Since the MAS process has an ultracentrifuge-like effect on

5

the sample, the 1H HR-MAS spectra would be expected to have a similar resolution to the 1H

6

solution NMR spectra of the supernatants if there were no contribution of the sediment to the

7

spectra. Therefore, relatively broader peaks of CBZ, SAC, and SDS in the nanosuspension

8

demonstrated the contribution of a semi-solid phase in the sediment to the linewidth of 1H HR-

9

MAS NMR spectra, by either superimposition or averaging of the dissolved and semi-solid

10

spectra. In the next section, this result is quantitatively discussed to understand the contribution

11

of each component to the semi-solid phase.

12 1

13

Figure 6.

H HR-MAS NMR spectra of (a) CBZ nanosuspension, (b) CBZ-SAC

14

nanosuspension, and (c) CBZ and SAC in the formulation vehicle of 0.5% (w/v) HPMC and

15

0.02% (w/v) SDS in water. Each spectrum was magnified individually to compare the spectra.

16


17


Page 18 of 29

1

Quantitative Analysis of the Dissolved and Semi-solid Components. The CBZ, SAC, HPMC,

2

and SDS signals detected in the solution 1H NMR spectra and the 1H HR-MAS NMR spectra

3

were quantified by peak integration to understand the molecular mobility distribution in detail.

4

Technically speaking, the concentrations calculated from the solution NMR signals in the

5

supernatant correspond to the dissolved components, while the HR-MAS NMR signals in the

6

nanosuspension reflect intermediate molecular mobility components such as the semi-solid phase

7

in addition to the dissolved components.26 Therefore, the semi-solid components can be

8

quantified by subtracting the solution-state NMR results for the supernatant from the HR-MAS

9

NMR results for the nanosuspension. The amounts of each component in µg/mL are listed in

10

Table 2. The concentrations of CBZ when CBZ dihydrate and CBZ-SAC were suspended in

11

water and the formulation vehicle are also shown in Table 3. Firstly, we focused on the dissolved

12

components. The concentrations of HPMC in the supernatants of the CBZ and CBZ-SAC

13

nanosuspensions were 4246 and 4016 µg/mL, respectively. The concentrations of SDS in the

14

supernatants of the CBZ and CBZ-SAC nanosuspensions were 112 and 50 µg/mL, respectively.

15

These results indicated that approximately 20% of the HPMC and 50%-75% of the SDS were

16

localized on the nanoparticles as a solid or semi-solid phase and, therefore, were removed by

17

ultracentrifugation, as the initial concentrations of HPMC and SDS in the formulation vehicle

18

were 5000 and 200 µg/mL, respectively. The concentration of CBZ in the supernatant of the CBZ

19

nanosuspension was 176 µg/mL, which was slightly higher than the apparent CBZ solubility of

20

CBZ dihydrate in the formulation vehicle (134 µg/mL). This discrepancy in the concentration

21

might be explained by the contribution of residual nanoparticles that were too small to be

22

removed by ultracentrifugation. On the other hand, the concentration of CBZ in the supernatant

23

of the CBZ-SAC nanosuspension was 201 µg/mL, which was obviously lower than the apparent


18

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1

CBZ solubility of CBZ-SAC in the formulation vehicle (370 µg/mL). This result supported the

2

hypothesis that HPMC and SDS participated in the nanoparticle structure as a solid phase,

3

because HPMC and/or SDS appeared to solubilize CBZ-SAC, as shown in Table 3. It was

4

speculated that the decrease in free HPMC and SDS molecules in the solution would lead to a

5

reduction in the solubilization efficiency of HPMC and/or SDS on CBZ-SAC.

6

Secondly, we considered the components in the semi-solid phase. The concentrations of SAC

7

and HPMC in the CBZ-SAC nanosuspensions were 2472 and 4672 µg/mL, respectively, which

8

were greater than those in the supernatants. As discussed previously in this section, the difference

9

between the concentrations determined from the supernatant and HR-MAS spectra should

10

correspond to the composition of the semi-solid phase; that is, 373 µg/mL SAC and 656 µg/mL

11

HPMC existed in the semi-solid phase in the CBZ-SAC nanosuspension. This result

12

quantitatively demonstrated, for the first time, an intermediate mobility phase in a

13

pharmaceutical nanosuspension. Unlike in the CBZ-SAC nanosuspension, the concentrations of

14

CBZ and SDS in both nanosuspensions, and of HPMC in the CBZ nanosuspension, were similar

15

to those in the supernatants, suggesting that the semi-solid phase of CBZ, SDS, and HPMC had

16

very limited contribution to the HR-MAS spectra because their molecular mobility in the semi-

17

solid phase was not in the HR-MAS sensitive region. This result indicated that those components

18

had lower molecular mobility than SAC and HPMC in the semi-solid phase of CBZ-SAC

19

nanosuspension, and thus, existed in a more rigid state than the semi-solid phase.

20

Finally, it is worth mentioning that concentrations of CBZ and SAC observed in the solution

21

and semi-solid phase were not equimolar, although the stoichiometry of CBZ and SAC in the

22

cocrystal structure was one-to-one. The excess of SAC observed in the CBZ-SAC

23

nanosuspension by solution and HR-MAS NMR indicated the dissociation of SAC from the


19


Page 20 of 29

1

cocrystal structure, followed by the potential isolation of solid CBZ driven by the low solubility

2

of CBZ. Nevertheless, single CBZ component that could potentially correspond to CBZ

3

dihydrate was not detected in the 13C CP/MAS NMR. Therefore, the existence of an undetectable

4

CBZ phase whose molecular mobility was too high to be detected in CP/MAS NMR and too low

5

to be observed in HR-MAS NMR was speculated. In the case of the piroxicam/poloxamer

6

nanosuspension reported by Hasegawa et al., an amorphous phase was detected in the piroxicam

7

nanoparticles using solid-state NMR, and was thought to interact with the poly(propylene oxide)

8

chains to maintain the polymer on the surface of the nanoparticle.24 The CBZ molecule, which

9

was not detected in any of the NMR measurements, could be in an amorphous state continuously

10

bridging the crystalline core and semi-solid interfacial structure.

11

Quantitative analysis demonstrated that some components were partially localized on the

12

surface of the nanoparticles and formed a semi-solid phase. In the semi-solid phase of the CBZ-

13

SAC nanosuspension, the molecular mobility of SAC and HPMC were relatively higher than

14

those of CBZ and SDS. CBZ formed an amorphous phase that was not detected either in the

15

CP/MAS or HR-MAS NMR spectra. The composition and molecular mobility of the surface

16

structure would play an important role in the stabilization of the nanoparticles, because it is the

17

surface structure which determines the physicochemical characteristics of the interface. The

18

surface structure of CBZ-SAC nanosuspension contained SAC and showed a relatively higher

19

molecular mobility compared to that of the CBZ nanosuspension. It was concluded that the ionic

20

compound SAC modified the surface structure, resulting in the different zeta potential of the

21

nanosuspension, and thus, its different physicochemical properties.17

22 23


20

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1

Table 2. Quantitative Values of Each Component in the Supernatants and Nanosuspensions

2

Determined from the Different NMR Measurements (n=3)

API

Sample

Method

CBZ

Supernatant

CBZ-SAC

Concentration (µg/mL, Mean ± S.D.) CBZ

SAC

HPMC

SDS

Solution

176 ± 2

-

4246 ± 89

112 ± 3

Nanosuspension

HR-MAS

172 ± 4

-

4373 ± 51

94 ± 3

Supernatant

Solution

201 ± 6

2099 ± 50

4016 ± 130

50 ± 2

Nanosuspension

HR-MAS

206 ± 8

2472 ± 39

4672 ± 159

47 ± 7

3

*Solution NMR of the supernatants represents dissolved components, while HR-MAS NMR of

4

the nanosuspensions indicates a total concentration of the semi-solid components plus the

5

dissolved components.

6

Table 3. Concentration of CBZ in Distilled Water and Distilled Water Containing 0.5% (w/v)

7

HPMC and 0.02% (w/v) SDS at 25°C Determined from HPLC Method (n=3) CBZ concentration (µg/mL, Mean ± S.D.)

API Water

0.5%HPMC + 0.02%SDS

CBZ dihydrate

134 ± 12

134 ± 9

CBZ-SAC

152 ± 22

370 ± 26

8 9

CONCLUSIONS. The detailed structures of the nanosuspensions were revealed by a

10

combination of NMR studies. Solid-state NMR measurements demonstrated that the solid phase

11

of the nanosuspensions consisted of crystalline API and solid HPMC. Solid HPMC was observed

12

even though HPMC was used as an aqueous solution, suggesting that intermolecular interactions

13

drove the solidification of the HPMC molecules during the wet milling process. The solution-

14

state NMR spectra of the nanosuspensions with and without ultracentrifugation pre-treatment

15

strongly suggested that the dissolved CBZ, SAC, and SDS molecules were in a fast equilibrium

16

with the low molecular mobility phase as a semi-solid phase. These experiments also suggested


21


Page 22 of 29

1

that the crystalline core particles were covered with a semi-solid phase composed of the API and

2

stabilizing agents. HR-MAS NMR measurements provided a direct evaluation method for the

3

semi-solid phase, which was not detected by either solid- or solution-state NMR. Quantitative

4

analysis revealed that the molecular mobility of SAC and HPMC in the semi-solid phase of the

5

CBZ-SAC nanosuspension were relatively higher than those of the other components. It was

6

concluded that the nanosuspensions were composed of crystalline API core particles surrounded

7

by a semi-solid phase consisting of the API and stabilizing agents. The semi-solid phase on the

8

nanoparticle surface was in equilibrium with the solution phase and prevented aggregation of the

9

nanoparticles by the steric hindrance and electrostatic repulsion caused by the stabilizing agents.

10

This research revealed the detailed structure and composition of the nanoparticles at the

11

molecular level. Understanding the molecular state of drug substances and stabilizing agents,

12

which determine the physicochemical properties of the nanoparticles, is critical for the efficient

13

development of nanosuspension formulations. We also expect that further investigation of

14

various nanosuspension formulations using the framework shown in this study will provide new

15

insights into nanoparticle structure.

16 17

ASSOCIATED CONTENT

18

Supporting Information. The following information is available in PDF file free of charge: 1H

19

spin-lattice relaxation times (T1H) of the solid materials (Table S1), 1H spin-lattice relaxation

20

times (T1H) of the compounds dissolved in D2O (Table S2), Physicochemical properties of the

21

nanosuspensions after

22

vehicle on the solution-state NMR peak linewidth (Figure S1).

13

C CP/MAS NMR measurements (Table S3), Impact of the formulation


22

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

AUTHOR INFORMATION

2

Corresponding Author

3

*E-mail: [email protected], Tel: +1-858-731-3551

4

Present Address

5

10410 Science Center Drive, San Diego, California, 92121, USA

6

Notes

7

The authors declare no competing financial interest.

8

ACKNOWLEDGMENT

9

We appreciate Junpei Takeda in Analytical Development of Takeda Pharmaceutical Company,

10

Ltd. for NMR measurement support. We also thank colleagues in Analytical Development of

11

Takeda Pharmaceutical Company, Ltd. for helpful discussion.

12

ABBREVIATIONS

13

CBZ, Carbamazepine; SAC, Saccharin; CBZ-SAC, carbamazepine-saccharin cocrystal; HPMC,

14

Hydroxypropyl

15

(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium; HMB, hexamethylbenzene; API, active

16

pharmaceutical ingredients; CP, cross polarization; MAS, magic angle spinning; TOSS, total

17

sideband suppression; HR, high resolution; PXRD, powder X-ray diffraction; PDI,

18

polydispersity index.

19

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methylcellulose;

SDS,

sodium

dodecyl

sulfate;

TSP-d4,

3-


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